5-substituted hydantoin racemase, DNA coding for the racemase, and processes for producing optically active amino acids

ABSTRACT

The present invention relates to a 5-substituted hydantoin racemase, which efficiently catalyzes racemization reactions at a high optimum temperature for racemization reactions, DNA coding for the racemase, and processes for producing optically active amino acids.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/863,245, filed on Jun. 9, 2004, which is a divisional of U.S.application Ser. No. 09/950,772, filed on Sep. 13, 2001 (now U.S. Pat.No. 6,815,195), which claims priority to: JP 2000-278571, filed on Sep.13, 2000 and JP 2001-65815, filed on Mar. 8, 2001.

FIELD OF THE INVENTION

The present invention relates to a 5-substituted hydantoin racemase,which efficiently catalyzes racemization reactions at a high optimumtemperature for racemization reactions, DNA coding for the racemase, andprocesses for producing optically active amino acids.

BACKGROUND OF THE INVENTION

The 5-substituted hydantoin racemase enzyme (hereinafter abbreviated toHRase) catalyzes a racemization reaction of an optically active5-substituted hydantoin compound, i.e., a D- or L-5-substitutedhydantoin compound.

The 5-substituted hydantoin compound undergoes a hydrolysis reactionwith hydantoinase (1) and Carbamyl amino acid hydrolase (2) to form anamino acid as shown in the following reaction scheme (I):

As shown in the reaction scheme above, the hydantoinase hydrolyzes the5-substituted hydantoin compound thereby forming N-carbamyl amino acid.Additionally, the N-carbamyl amino acid hydrolase hydrolyzes N-carbamylamino acid thereby forming an optically active amino acid. These enzymesshould be optically selective.

Methods of using a microbial enzyme system and combining a bacterialenzyme system with a chemical reaction system have been previouslydescribed. Production of optically amino acids from 5-substitutedhydantoin compounds is important in the production of pharmaceuticalpreparations, chemical industry products, food additives and othersimilar articles or products.

The optico-selective hydrolysis should enable the efficient racemizationof one enantiomer to another enantiomer, the second enantiomer canfurther serve as a substrate, for example see the above reaction scheme.Furthermore, this conversion can be performed using microbial enzymes ormicrobial enzymes combined with a chemical reaction system. Underneutral conditions where the enzyme system is active, the racemizationof the optically active 5-substituted hydantoin compound, not serving asthe substrate, is very low. Therefore, the racemization becomesrate-determining and resulting in poor conversion into the opticallyactive amino acid.

Accordingly, for the purpose of racemization of the optically active5-substituted hydantoin compound under neutral conditions, HRase wassearched for, and HRases derived from microorganisms of the genusArthrobacter (Japanese Patent Application Laid-Open (JP-A) No.62-122591; Ann. N.Y. Acad. Sci., 672, 478; Japanese Patent ApplicationLaid-Open (JP-A) No. 6-343462) and microorganisms of the genusPseudomonas (Japanese Patent Application Laid-Open (JP-A) No. 4-271784;J. Bacteriol., 174, 7989 (1992)) have been reported. The optimumreaction temperatures of the previously reported HRases derived frommicroorganisms are 37° C. for the enzyme derived from Arthrobacter sp.DSM-3747 (Ann. N.Y. Acad. Sci., 672, 478), 10 to 50° C. for the enzymederived from Arthrobacter sp. DK200 (Japanese Patent ApplicationLaid-Open (JP-A) No. 62-122591), and 45° C. for the enzyme derived fromPseudomonas sp. NS671.

Generally, when the working optimum temperature of an enzyme isincreased, the industrial utility value of the enzyme is also increased.That is, if the reaction temperature can be increased, the reaction ratecan be also increased, which results not only in efficient progress ofthe desired reaction but also in a reduction in the risk ofcontamination of the reaction solution with microorganisms during thereaction. This results in several advantages including easy process andquality control.

As described above, HRase having a higher optimum temperature for thereaction is useful from the viewpoint of industrial applicability.However, the upper limit of the working optimum temperatures of thepreviously reported HRases is 50° C. and an HRase having a higherworking optimum temperature is desired to achieve efficient progress ofthe racemization reaction and to reduce the risk of contamination of thereaction solution with microorganisms.

SUMMARY OF THE INVENTION

It is an object of the present invention to isolate a novel HRase havinga higher reaction optimum temperature than conventional and to provide aprocess for producing an optically active amino acid by use of theenzyme.

As a result of extensive study in view of the problem described above,the present inventors found a novel HRase having a desired higherreaction optimum temperature is present in a Microbacteriummicroorganism, thus arriving at completion of the present invention.

One object of the present invention, is providing a new process adjuvantfor improving the racemization reaction of 5-substituted hydantoincompounds and optically active amino acids.

Another object of the invention is to provide a nucleotide sequenceencoding a polypeptide which has HRase activity. One embodiment of sucha sequence is the nucleotide sequence of SEQ ID NO: 1. Other embodimentsinclude nucleotide sequences that are complimentary to the sequencesdescribed herein, those sequences that are 70%, 80% and/or 90% identicalto the sequences described herein and/or those sequences that hybridizeunder stringent conditions to the sequences described herein.

A further object of the invention is a method of making HRase or anisolated polypeptide having a HRase activity, as well as use of suchisolated polypeptides in the production of amino acids. One embodimentof such a polypeptide is the polypeptide having the amino acid sequenceof SEQ ID NO: 2. Other embodiments are this polypeptide are those thatare 70%, 80% and/or 90% identical to the amino acid sequences describedherein. Another embodiment of the polypeptide is that which has racemaseactivity with an optimal working pH of from about 7 to about 9 and anoptimal working temperature of from about 50 to about 60° C.

Other objects of the invention include methods of detecting nucleic acidsequences homologous to the sequences described herein, particularlynucleic acid sequences encoding polypeptides that have HRase activity,and methods of making nucleic acids encoding such polypeptides.

Another object of the present invention is to provide methods ofpreparing optically active hydantoin compounds by contacting a5-substituted hydantoin compound with the HRase described herein.

Another object of the present invention is to provide methods ofpreparing an N-carbamyl amino acid by contacting a 5-susbstitutedhydantoin compound with the HRase described herein and an enzyme thatwill hydrolyze the 5-substituted hydantoin compound in an opticallyselective manner. One embodiment of an enzyme that will hydrolyze the5-substituted hydantoin compound is the Hydantoinase described herein.The Hydantoinase may have the amino acid sequence of SEQ ID NO:4 oramino acid sequences with substantial identity to SEQ ID NO:4 and havingthe hydantoinase activity.

Another object of the present invention is to provide methods ofproducing optically active amino acids by contacting a 5-substitutedhydantoin compound with the HRase described herein, an enzyme that willhydrolyze the 5-substituted hydantoin compound in an optically selectivemanner, and an enzyme that will hydrolyze a N-carbamyl amino acid in anoptically selective manner. One embodiment of an enzyme that willhydrolyze the 5-substituted hydantoin compound is the Hydantoinasedescribed herein. The Hydantoinase may have the amino acid sequence ofSEQ ID NO:4 or amino acid sequences with substantial identity to SEQ IDNO:4 and having the hydantoinase activity. One embodiment of the enzymethat will hydrolyze the N-carbamyl amino acid is the carbamyl amino acidhydrolase described herein. The carbamyl amino acid hydrolase may havethe amino acid sequence of SEQ ID NO:6 or amino acid sequences withsubstantial identity to SEQ ID NO:6 and having the hydrolase activity.

The above objects highlight certain aspects of the invention. Additionalobjects, aspects and embodiments of the invention are found in thefollowing detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing showing a group of structural genes coding forhydantoin racemase, hydantoinase and N-carbamyl-L-amino acid hydrolase.

FIG. 2 is a flow chart showing a process for producing the hydantoinracemase of the present invention.

FIG. 3 is a drawing showing the optimum pH curve of the hydantoinracemase of the present invention.

FIG. 4 is a drawing showing the pH stability curve of the hydantoinracemase of the present invention.

FIG. 5 is a drawing showing the optimum temperature curve of thehydantoin racemase of the present invention.

FIG. 6 is a drawing showing the temperature stability curve of thehydantoin racemase of the present invention.

FIG. 7 is a drawing showing the time course of racemization of BH by thehydantoin racemase of the present invention. Where, ◯: Substrate D-BH(HRase added), Δ: Substrate D-BH (HRase not added), ●: Substrate L-BH(HRase added), ▴: Substrate L-BH (HRase not added).

FIG. 8 is a drawing showing the time course of formation of D-Phe.Where, □: Substrate D-BH (HRase not added), ◯: Substrate DL-BH (HRasenot added), Δ: Substrate L-BH (HRase not added), ▪: Substrate DL-BH(HRase added), ▴: Substrate L-BH (HRase added).

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art of molecular biology. Although methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present invention, suitable methods and materials aredescribed herein. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and are not intended to be limiting.

Reference is made to standard textbooks of molecular biology thatcontain definitions and methods and means for carrying out basictechniques, encompassed by the present invention. See, for example,Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, New York (1982) and Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York(1989) and the various references cited therein.

[I] HRase

Among Flavobacterium microorganisms, a bacterial strainoptico-selectively hydrolyzing DL-5-indolyl methyl hydantoin to form itscorresponding L-tryptophan is reported. When this microbial strain(Flavobacterium sp. AJ3912 (FERM-P3133) ) forming L-tryptophan is usedin the reaction of forming L-tryptophan from DL-5-indolyl methylhydantoin as the substrate, the molar yield of L-tryptophan formedreaches 80% or more (Agric. Biol. Chem., 51, 363 (1987)).

Since spontaneous racemization of optically active indolyl methylhydantoin rarely occurs under neutral conditions where the enzymereaction is carried out, it was presumed that a novel HRase is presentin the microbial strain. On the basis of this presumption, the presentinventors purified and isolated a HRase from a cultured microorganism.This novel enzymes was found to have a desired high optimum reactiontemperature.

Flavobacterium sp. AJ3912 (FERM-P3133) was initially deposited asFlavobacterium sp. AJ3912 (FERM-P3133) on Jun. 27, 1975 with theNational Institute of Bioscience and Human-Technology, the Agency ofIndustrial Science of Technology, the Ministry of International Tradeand Industry, Japan, which was later reclassified as a Microbacteriumliquefaciens microorganism. Thus, this microorganism is deposited asMicrobacterium liquefaciens AJ3912 (FERM-P3133) with the NationalInstitute of Bioscience and Human-Technology, the Agency of IndustrialScience of Technology, the Ministry of International Trade and Industry.

The physiological properties of Microbacterium liquefaciens AJ3912(FERM-P3133) were examined in light of a microbial classification book,Berjey's Manual of Determinative Bacteriology, Vol. 1 (9th edition,1994, William & Wilkins Publishing Company), and the test results areshown in Table 1. TABLE 1 Results of re-identification of Microbacteriumliquefaciens AJ3912 Gram stainability positive Motility none Nitratereduction − Pyrimidinase − Pyridonyl allyl amidase − Alkaliphosphatase + β-Glucuronidase − β-Galactosidase + α-Glucosidase +N-Acetyl-β-glucosaminidase + Aesculin (glucosidase) + Urease − Gelatinliquefaction + Fermentability of hydrocarbons glucose − ribose − xylose− mannitol − maltose − lactose − white sugar − glycogen − Anaerobicgrowth − Casein hydrolyzability +

The present inventors purified HRase derived from Microbacteriumliquefaciens AJ3912, and the amino acid sequence of the HRase wasdetermined. From this amino acid sequence an approximately 30-bp DNAmolecule was synthesized and used as to probe a Microbacteriumchromosomal library. This resulted in the successful isolation afull-length DNA that codes for the Microbacterium DRase.

Further, t he present inventors predicted that a nucleotide sequencedownstream from the HRase gene, which was simultaneously obtained inisolation of the HRase is a part of a hydantoinase (HHase) gene. Thus,this DNA fragment was amplified by PCR and utilized as a probe tosuccessfully isolate the Hhase gene. The present inventors alsopredicted that a nucleotide sequence downstream from the HHase gene,which was simultaneously obtained in isolation of the HHase gene, is apart of an N-carbamyl amino acid hydrolase (CHase) gene. Thus, this DNAfragment was amplified by PCR and utilized as a probe to successfullyisolate the Chase gene.

As shown in FIG. 1, the HRase gene of the present invention isconsidered to form an operon together with the HHase gene and CHasegene. In FIG. 1, (1) is an EcoRI/PstI fragment, (2) is a KpnI/SacIfragment, and (3) is a BglII fragment.

In the accompanying sequence listing of the present invention, the DNAcoding for the HRase of the present invention is shown in SEQ ID NO:1,the DNA coding for the HHase is shown in SEQ ID NO:3, and the DNA codingfor the CHase is shown in SEQ ID NO:5, as determined by the methoddescribed above. Further, DNA coding for a group of structural genesincluding the HRase gene, HHase gene and CHase gene is set forth in SEQID NO:7. In the nucleotide sequence set forth in SEQ ID NO:7, thenucleotide residues 1 to 708 code for the HRase of the presentinvention, the nucleotide residues 729 to 2105 code for the HHase, andthe nucleotide residues 2105 to 3340 code for the CHase.

These DNAs were those isolated from the chromosomal DNA inMicrobacterium liquefaciens AJ3912 and code for proteins related to theproduction of L-amino acids.

Further, the amino acid sequence of the HRase encoded by the nucleotidesequence in SEQ ID NO:1 in the Sequence Listing is set forth in SEQ IDNO:2 in the Sequence Listing, the amino acid sequence of the HHaseencoded by the nucleotide sequence in SEQ ID NO:3 in the SequenceListing is set forth in SEQ ID NO:4 in the Sequence Listing, and theamino acid sequence of the CHase encoded by the nucleotide sequence inSEQ ID NO:5 in the Sequence Listing is set forth in SEQ ID NO:6 in theSequence Listing.

As shown in the reaction scheme below, the HRase having the sequence ofSEQ ID NO:2 g, the Hhase having the sequence of SEQ ID NO:4 and theCHase having the sequeence of SEQ ID NO:6 catalyze the reaction ofcoverting a 5-substituted hydantoin (5-(4-hydroxylbenzyl)hydantoin) toan optically active amino acid (e.g., L-tyrosine) as shown in thereaction scheme (II) below:

(1) DNA Coding for the HRase Enzyme

As described above, the present HRase gene having the nucleotidesequence of SEQ ID NO:1 was isolated from the chromosomal DNA ofMicrobacterium liquefaciens AJ3912. The amino acid sequence of SEQ IDNO:2 and the nucleotide sequence of SEQ ID NO: 1 exhibit 48% homology inthe amino acid sequence and 49% homology in the nucleotide sequence to aknown HRase derived from a Pseudomonas microorganism (J. Bacteriol. 174,962 (1992)).

The method of acquiring DNA coding for the HRase from the microorganismof the genus Microbacterium is described below.

First, the amino acid sequence of the purified HRase is determined. Theamino acid sequence can be determined by use of the Edman method (Edman,P., Acta Chem. Scand. 4, 227 (1950)). Alternatively, the amino acidsequence can be determined by use of a sequencer manufactured by AppliedBiosystems. By determining the amino acid sequence of 30 residues fromthe N-terminus in the HRase of the present invention derived from theMicrobacterium microorganism, the sequence shown in SEQ ID NO:8 wasdetermined.

Based on this amino acid sequence, the nucleotide sequence of DNA codingfor the same can be deduced using the universal codons known in the art.

Based on the deduced nucleotide sequence, a DNA molecule ofapproximately 30-bp is synthesized. The method of synthesizing the DNAmolecule is disclosed in Tetrahedron Letters, 22, 1859 (1981).Alternatively, the DNA molecule can be synthesized by use of asynthesizer manufactured by Applied Biosystems. The DNA molecule can beused as a probe so that full-length DNA coding for the HRase is isolatedfrom a Microbacterium chromosomal gene library. Alternatively, the DNAmolecule can be used as a primer by which DNA coding for the HRase isamplified by PCR. However, if the amplified DNA amplified does notcontain a full-length coding DNA, the amplified DNA may be used as aprobe to obtain the full-length coding DNA from a Microbacteriumchromosomal gene library.

PCR amplification is known in the art and is described, for example, inWhite, T. J. et al., in Trends Genet. 5, 185 (1989). The method ofpreparing chromosomal DNA and the method of isolating the desired DNAmolecule from a gene library by use of the above DNA molecule as a probeare described are known in the art and described in, for example,Molecular Cloning, 2nd edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, New York(1989).

The method of determining the nucleotide sequence of the isolated DNAcoding for the HRase is generally known in the art and described, forexample, in A Practical Guide to Molecular Cloning, John Wiley & Sons,Inc., New York (1985). Alternatively, the nucleotide sequence can bedetermined by use of a DNA sequencer manufactured by Applied Biosystems.The DNA coding for the Microbacterium HRase is shown in SEQ ID NO:1.

The DNA molecules encoding Microbacterium HRase is not limited to theDNA set forth in SEQ ID NO:l in the Sequence Listing. This is becausethe nucleotide sequence must be varied depending on the species andstrain of each Microbacterium microorganism.

As a matter of course, the DNA of the present invention encompasses notonly the isolated DNA coding for the HRase but also HRase-coding DNAwhich can be prepared by artificially mutating the HRase-coding DNAisolated from the chromosomal DNA in the microorganism of the genusMicrobacterium. For example, site-specific mutagenesis methods as knownin the art and described, for example, in Methods in Enzymology, 154(1987).

The DNA of the present invention also encompasses DNA having anucleotide sequence hybridizing under stringent conditions with thenucleotide sequence set forth in SEQ ID NO:1 in the Sequence Listing andcoding for a protein having a 5-substituted hydantoin racemase activity.As used herein, “stringent conditions” refer to those conditions underwhich a specific hybrid is formed whereas an unspecific hybrid is notformed. These conditions will vary depending on the nature, length, basecontent, etc. of the DNA. Specificity is typically the function ofpost-hybridization washes, the critical factors being the ionic strengthand temperature of the final wash solution. For DNA—DNA hybrids, theT_(m) can be approximated from the equation of Meinkoth and Wahl, Anal.Biochem., 138:267-284 (1984): T_(m)=81.5° C.+16.6 (log M)+0.41 (%GC)-0.61 (% form)-500/L; where M is the molarity of monovalent cations,% GC is the percentage of guanosine and cytosine nucleotides in the DNA,% form is the percentage of formamide in the hybridization solution, andL is the length of the hybrid in base pairs. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of acomplementary target sequence hybridizes to a perfectly matched probe.T_(m) is reduced by about 1° C. for each 1% of mismatching; thus, T_(m),hybridization and/or wash conditions can be adjusted to hybridize tosequences of the desired identity. For example, if sequences withapproximately 90% identity are sought, the T_(m) can be decreased 10° C.Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (T_(m)) for the specific sequence and itscomplement at a defined ionic strength and pH. However, severelystringent conditions can utilize a hybridization and/or wash at 1, 2, 3,or 4° C. lower than the thermal melting point (T_(m)) ; moderatelystringent conditions can utilize a hybridization and/or wash at 6, 7, 8,9, or 10° C. lower than the thermal melting point (T_(m)); lowstringency conditions can utilize a hybridization and/or wash at 11, 12,13, 14, 15, or 20° C. lower than the thermal melting point (T_(m)).Using the equation, hybridization and wash compositions, and desiredT_(m), those of ordinary skill will understand that variations in thestringency of hybridization and/or wash solutions are inherentlydescribed. If the desired degree of mismatching results in a T_(m) ofless than 45° C. (aqueous solution) or 32° C. (formamide solution) it ispreferred to increase the SSC concentration so that a higher temperaturecan be used. An extensive guide to the hybridization of nucleic acids isfound in Tijssen, Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, New York (1993); and Current Protocols inMolecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishingand Wiley-Interscience, New York (1995).

For example, stringent conditions will allow hybridization between DNAmolecules having higher homology, for example 50% or more, morepreferably 70%, more preferably 80% or more and most preferably 90% ormore. Under these stringent conditions DNA molecules having lowerhomology would not hybridize with each other, or to those conditionsunder which hybridization occurs under usual washing conditions inSouthern hybridization, that is, at 60° C., a salt concentration of1×SSC and 0.1% SDS, preferably at 60° C., 0.1×SSC and 0.1% SDS and morepreferably at 65° C., 0.1×SSC and 0.1% SDS.

The “5-substituted hydantoin racemase activity” may be any activity ofracemizing a 5-substituted hydantoin compound. However, the nucleotidesequence hybridizing under stringent conditions with the nucleotidesequence set forth in SEQ ID NO:1 in the Sequence Listing preferablymaintain about at least half of the enzyme activity of a protein havingthe amino acid sequence set forth in SEQ ID NO:2 in the Sequence Listingunder reaction conditions of 50° C. and pH 8 and as described herein.

The DNA of the present invention also encompasses DNA coding forsubstantially the same protein as the HRase encoded by the DNA set forthin SEQ ID NO:1. That is, the DNA of the present invention encompassesDNA coding for a protein having a 5-substituted hydantoin racemaseactivity and having the following amino acid sequence:

(a) the amino acid sequence set forth in SEQ ID NO:2 in the SequenceListing, or

(b) an amino acid sequence wherein in the amino acid sequence set forthin SEQ ID NO:2 in the Sequence Listing, one or more amino acid residueshave been replaced, deleted, inserted, added or inverted. Here, thenumber of “one or more amino acid residues” is in such a range that thestereostructure of the proteins of amino acid residues or the5-substituted hydantoin racemase activity is not significantlydeteriorated; specifically, the number of such amino acids is 2 to 50,preferably 2 to 30 and more preferably 2 to 10. However, the amino acidsequence (b) wherein in the amino acid sequence set forth in SEQ ID NO:2in the Sequence Listing, one or more amino acid residues have beenreplaced, deleted, inserted, added or inverted, preferably has about atleast half of the enzyme activity of a protein having the amino acidsequence set forth in SEQ ID NO:2 under reaction conditions of 50° C.and pH 8. Furthermore, the invention also encompasses DNA encodingproteins having the aforementioned activity and which aresubstantionally identical to SEQ ID NO:2, preferably at least 70%, 80%,90% or 95% identical.

(2) Properties of HRase

As revealed by isolation and analysis of the above-described gene, theHRase of the present invention has the amino acid sequence set forth inSEQ ID NO:2. However, the present invention also encompasses a proteinhaving a 5-substituted hydantoin racemase activity and having an aminoacid sequence wherein in the amino acid sequence set forth in SEQ IDNO:2 in the Sequence Listing, one or more amino acid residues have beenreplaced, deleted, inserted, added or inversed. That is, the HRase ofthe present invention is a protein having a 5-substituted hydantoinracemase activity and having the following amino acid sequence (a) or(b):

(a) the amino acid sequence of SEQ ID NO:2, and

(b) an amino acid sequence wherein in the amino acid sequence of SEQ IDNO:2, one or more amino acid residues have been replaced, deleted,inserted, added or inverted.

The terms “one or more amino acid residues” and “5-substituted hydantoinracemase activity” have the same meanings as defined in item (1) DNAcoding for HRase. Also included within the scope of the presentinvention are those proteins that have the herein described racemizationactivity and have 70%, preferably 80%,-more preferably 90% or 95%identity with the amino acid sequence of SEQ ID NO:2.

Homology, sequence similarity or sequence identity of nucleotide oramino acid sequences may be determined conventionally by using knownsoftware or computer programs such as the BestFit or Gap pairwisecomparison programs (GCG Wisconsin Package, Genetics Computer Group, 575Science Drive, Madison, Wis. 53711). BestFit uses the local homologyalgorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of identity or similaritybetween two sequences. Gap performs global alignments: all of onesequence with all of another similar sequence using the method ofNeedleman and Wunsch, J. Mol. Biol. 48:443-453 (1970). When using asequence alignment program such as BestFit, to determine the degree ofsequence homology, similarity or identity, the default setting may beused, or an appropriate scoring matrix may be selected to optimizeidentity, similarity or homology scores. Similarly, when using a programsuch as BestFit to determine sequence identity, similarity or homologybetween two different amino acid sequences, the default settings may beused, or an appropriate scoring matrix, such as blosum45 or blosum80,may be selected to optimize identity, similarity or homology scores.

The HRase of the present invention catalyzes the racemization reactionof a 5-substituted hydantoin compound.

Measurement of the HRase activity of the HRase of the present inventioncan be carried out by measuring a change in the optical rotation as thedegree of racemization of the optically active D- or L-5-substitutedhydantoin compound as the substrate or by high performance liquidchromatography (HPLC) using an optical resolution column.

Specifically, a reaction solution containing 120 mg/dl L- or D-benzylhydantoin (BH), 50 mM dibasic potassium phosphate buffer (KPB) (pH 8.0),5 mM dithiothreitol (DTT) and an HRase enzyme solution was incubated at37° C. for 30 minutes, and the reaction was stopped by adding a 9-foldexcess volume of 1.1 mM CuSO₄ and 11.1 mM H₃PO₄ solution. Theprecipitates were removed by centrifugation at 20,000 g×10 minutes, andthe amount of racemized BH was quantitatively determined by HPLC toestimate the racemization activity. The enzyme activity causingracemization of 1 μmol BH per minute under these conditions was definedas 1 U of the enzyme activity.

The quantitative analysis of optically active BH by analysis in HPLC wasconducted by use of Daicel Chemical CHIRALPAK WH 0.46 cmφ×25 cm. Theanalysis conditions are as follows:

Mobile phase: 5% (v/v) methanol, 1 mM CUSO₄

Column temperature: 50° C.

Flow rate: 1.5 ml/min.

Detection: UV₂₁₀

Under these conditions, D-BH (retention time: 4.2 minutes) and L-BH(retention time: 5.3 minutes) were eluted.

Then, the enzymatic chemical properties of the HRase of the presentinvention measured by the above analysis method are described below.

The working optimum temperature of the HRase of the present inventionlies in a higher temperature range than that of the previously reportedHRases, and thus the present enzyme is characterized by the ability tocatalyze racemization reactions efficiently. That is, the HRase of thepresent invention are characterized as having a working optimumtemperature, though varying at a certain degree due to a difference inthe amino acid sequence, of 50° C. or more, preferably 52° C. or more,more preferably 55° C. or more. The upper limit of the working optimumtemperature is not particularly limited, but in consideration of thethermostability of HRase, the upper limit is preferably 60° C. or less.The “working optimum temperature” in the present specification means thetemperature at which the maximum activity is exhibited under at pH 8.

To determine the range of the working optimum pH, the reaction wascarried out for 30 minutes under the condition of 40° C. The optimum pHwas determined to be from approximately 7 to approximately 9.

The pH stability after treatment at each pH for 30 minutes under thecondition of 0° C. was examined. The pH stability was determined to befrom approximately 6 to approximately 9.

The temperature stability after heating treatment for 30 minutes underthe condition of pH 8.0 was examined and was determined to beapproximately 40° C. or less. The HRase of the present invention isstrongly inhibited by NEM (N-ethyl maleimide), copper ion, or IAA(monoiodoacetatic acid) but minimally inhibited by EDTA(ethylenediaminetetraacetic acid). The HRase of the invention isactivated by adding DTT (dithiothreitol). The Molecular weight of theHRase of the present invention: a) about 107,000 determined by gelfiltration, or b) about 27,000 determined on a SDS-PAGE. Based on theseresults it is believed that the HRase of the present invention is atetramer, where each subunit of the tetramer has a molecular weight ofapproximately 27,000.

(3) Process for producing HRase and a protein having a 5-substitutedhydantoin racemase activity

The process for producing the HRase of the invention and a proteinhaving a 5-substituted hydantoin racemase activity includes two methods,that is, (i) a process which comprises culturing a microorganism to formand accumulate HRase, and (ii) a process of transforming a microorganismwith a gene encoding HRase or a protein having a 5-substituted hydantoinracemase activity by recombinant DNA technology and culturing thetransformant to form and accumulate the protein.

(i) Process of formation and accumulation by culturing a microorganism

The microorganim culturing methods preferably emplopy a MicrobacteriumHRase. Preferable microorganisms include Microbacterium liquefaciensAJ3912 (FERM-P3133).

The form of culture of Microbacterium microorganisms may be eitherliquid culture or solid culture, but an industrially advantageous methodis a submerged aeration shake culture method. Nutrient sources in theliquid medium can make use of carbon sources, nitrogen sources,inorganic salts and other trace nutrient sources, which are usually usedin culture of microorganisms. Any nutrient sources can be used insofaras they can be utilized by the bacterial strain cultured.

As the aeration conditions, aerobic conditions are preferably used. Theculture temperature may be in any range in which the microorganism growsand produces HRase. Accordingly, there are no strict conditions, but theculture temperature is usually 10 to 50° C., preferably 30 to 40° C. Theculture time is varied depending on other culture conditions. Forexample, the microorganism may be cultured until most of the HRase isproduced, and the culture time is usually 5 hours to 7 days, preferably10 hours to 3 days or thereabout.

Following culturing the enzyme can be obtained by collecting themicroorganisms by centrifugation (e.g., 10,000×g, 10 minutes). Becausethe enzyme is present in the microorganism, this microorganism may bedisrupted or lysed to faciliate solublization of the enzyme. Methods ofdisrupting cells are known in the art and include, for example,sonication, French press, glass beads, treatment with egg white lysozymeor peptidase enzymes, or suitable combinations of two or more of thesemethods.

To purify the HRase, the solubilized enzyme is used as the startingmaterial, and if undisrupted or non-lyzed microbial residues arepresent, it is advantageous to centrifuge the solubilized solution againto remove the precipitated residues.

For purification of the enzyme, enzyme purification methods known in theart can be employed. Such methods include, for example, salting-out withammonium sulfate, gel filtration, ion-exchange chromatography,hydrophobic chromatography, and other types of chromatography orpurification methods. As a result, a fraction containing HRase of higherspecific activity can be obtained.

(ii) Recombinant DNA Methods

The enzymes of the present invention can be advantageously preparedusing recombinant DNA technology commonly employed in the art tofaciliate scaling up the amount of enzyme produced by a microorganismand thus the amount of active enzyme obtained from the microorganismculturing methods described herein. An example of such methods is shownin FIG. 2 is a flow chart of the process for producing the HRase of thepresent invention.

First, the DNA coding for the HRase of the present invention is prepared(step S1).

Then, the prepared DNA is ligated to a vector DNA to prepare arecombinant DNA (step S2), and cells are transformed with therecombinant DNA, to prepare a transformant (step S3). Subsequently, thetransformant is cultured in a medium to form and accumulate HRase in themedium and/or the cells (step S4).

Thereafter, the enzyme is recovered and purified in step S5.

Further, the purified HRase produced in step S5 or the medium containingthe HRase in step S4 can be used in the synthesis of large amounts ofoptically active amino acids (step S6).

The DNA ligated to the vector DNA may be any DNA capable of expressingthe HRase of the present invention. The HRase encoding DNAs ligated intothe vector DNA include any of the DNA molecules described herein.

When the protein production is scaled up to yield large quantities ofprotein, the protein may aggregate in the transformant to form a proteininclusion body. The formation of protein inclusion bodies provides theadvantage of protecting the protein in the microorganism fromproteolyitc digestion in the cell and faciliates simpler purificationfollowing centrifugation and cell disruption as described above. Toobtain the active protein from the protein inclusion body, a series ofprocedures such as solubilization and activity restoration etc. arenecessary, and thus the procedures are more complex than in directproduction of the active protein. However, if a protein exerting aninfluence on growth of the microorganism is produced in a large amountin the microorganism, the influence can be suppressed by accumulatingthe protein as an inert inclusion body in the microorganism.

The protein inclusion body may be solubilized with a protein denaturantand renatured by removing the denaturant, for example, by dialysis.Following renaturation the activity of the protein is restored, asdescribed for the restoration of the activity of human interleukin-2(Japanese Patent Application Laid-Open (JP-A) No. 61-257931).

The method of producing a large amount of the desired protein as aninclusion body includes not only a method of expressing the desiredprotein solely under the control of a strong promoter but may alsoinclude expressing the protein as a fusion protein to facilatepurification. Preferably, when preparing the fusion protein a sequencerecognized by a proteolytic enzyme is provided between the HRase and thepeptide or polypeptide to which is fused. This proteolytic enzymesequence facilitates separation of the HRase from the fusion peptide orpolypeptide after purification. Examples of such proteolytic enzymesblood coagulation factor Xa or kallikrein whose recognition sequence isa sequence not present in HRase.

The host cells to be transformed for producing a large amount of theprotein by recombinant DNA technology include microbial cells,Actinomyces cells, yeast cells, fungal cells, plant cells, animal cellsetc., but in general Escherichia coli is preferably used. This isbecause there are a large number of techniques for a large production ofproteins by use of Escherichia coli. As the promoter expressing DNAcoding for HRase, a promoter ordinarily used in production of aheterogeneous protein in E. coli can be used or other promoters asappropriate to express the protein in the specific cell employed forexpression. Examples of such promoters include strong promoters such asthe T7 promoter, trp promoter, lac promoter, tac promoter, PL promoterand the like.

To produce HRase as a fusion protein inclusion body, a gene coding foranother protein, preferably a hydrophilic peptide, is ligated to aregion upstream or downstream of the HRase gene to produce afusion-protein gene. The gene coding for another protein may be any genewhich increases the amount of a fusion protein accumulated and raisesthe solubility of the fusion protein after denaturation and restoration,and for example, T7 gene 10, β-galactosidase gene, dehydrofolatereductase gene, interferon γ gene, interleukin-2 gene, prochymosin genemay be used.

When ligating the two genes together, the coding readings should be keptin-frame to insure appropriate translation of the fusion protein. Theymay be ligated at suitable restriction enzyme sites, or synthetic DNA ofsuitable sequence may be utilized.

To increase the amount of the fusion protein produced, a terminator thatis a transcription termination sequence is preferably ligated to adownstream region of the fusion-protein gene. Examples of suchterminators include T7 terminator, fd phage terminator, T4 terminator, aterminator for tetracycline resistance gene, a terminator for E. colitrp A gene, and similar terminators as known to the skilled artisan.

Vectors for introducing the HRase gene or HRase fusion protein into E.coli are preferably multicopy vectors. Examples of such vectors includesplasmids having an origin of replication derived from Col E1: pUC seriesplasmids, pBR322 series plasmids or derivatives thereof. The“derivatives thereof” are those obtained by modifying plasmids byreplacement, deletion, insertion, addition or inversion of somenucleotides. The modification referred to here includes modification bynatural mutation or by mutation treatment with a mutagen or UVirradiation.

To select the transformant, the vector preferably has a marker or drugresistance genes known in the art. Examples of such drug resistancegenes include ampicillin resistance gene, kanamycin resistance gene,tetracyline resitance gene and the similar genes. As such plasmids,expression vectors having a strong promoter are commercially available(pUC series (Takara Shuzo Co., Ltd.), pPROK series (Clontech), pKK233-2(Clontech) etc.).

A DNA fragment having a promoter, a gene coding for HRase or a fusionprotein of HRase with another protein and a terminator ligated in thisorder is then ligated to a vector DNA to give a recombinant DNA.

The recombinant DNA is used to transform E. coli or other microorganismand the HRase or a fusion protein of HRase with another protein isexpressed and produced upon culturing the transformed cell.

Although the host to be transformed can be any strain ordinarily usedfor expressing a heterogeneous gene, Escherichia coli JM109,particularly Escherichia coli JM109 (DE3) is prefered. The method forconducting transformation and the method for selecting the transformantare described in, for example, Molecular Cloning, 2nd edition, ColdSpring Harbor Press (1989).

The production medium used may be a medium such as M9-casamino acidmedium, LB medium etc. which is usually used for culturing E. coli.Other appropriate media depending on the cell being cultured can beformulated as known in the art. Further, culture conditions andproduction-inducing conditions are selected suitably depending on amarker and a promoter in the vector used, the type of hostmicroorganism, and other factors that the skilled artisan wouldrecognize.

To recover HRase or a HRase fusion protein the following may beemployed. If HRase or its fusion protein has been solubilized, themicroorganism is recovered, disrupted or lyzed and the resulting crudeenzyme solution can be used. As necessary, HRase or its fusion proteincan be used in a pure form after purification as described herein. Inthis case, a method of purification by use of an antibody against HRaseor its fusion protein can also be used. Methods of making antibodiesreactive against HRase are known in the art and are described in, forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, New York (1989).

When the protein inclusion body is formed, it is solubilized with adenaturant. The inclusion body may be solubilized together withmicrobial proteins, but in consideration of the subsequent purificationprocedures, it is preferable that the inclusion body is removed andsolubilized. The inclusion body can be recovered from the microorganismby a method known in the art. For example, the microorganism isdisrupted and centrifuged etc. to recover the inclusion body. Thedenaturant for solubilizing the protein inclusion body includesguanidine hydrochloride (e.g., 6 M, pH 5 to 8) and urea (e.g. 8 M).

By removing the denaturant by dialysis, the activity of HRase isobtained. The dialysis solution used in dialysis may make use ofTris-HCl buffer or phosphate buffer, and a prefered concentration is 20mM to 0.5 M, and a prefered pH is 5 to 8.

The concentration of the protein in the regeneration step is preferablylimited to about 500 μg/ml or less. To prevent self-crosslinkage of theregenerated HRase, the dialysis temperature is preferably 5° C. or less.Further, the method of removing the denaturant includes a dilutionmethod and an ultrafiltration method besides the dialysis method, andusing any method, restoration of the activity can be expected.

If the DNA set forth in SEQ ID NO:1 in the Sequence Listing is used asDNA coding for HRase, HRase having the amino acid sequence set forth inSEQ ID NO:2 is produced.

[II] Process for Producing an Optically Active Amino Acid by HRase

The HRase of the present invention used in the reaction of producing anoptically active amino acid includes a protein having a hydantoinracemase activity and has an amino acid sequence as described herein.

As the HRase, it is possible to use (1) HRase obtained by culturing aMicrobacterium microorganism or (2) by preparing an HRase-producingtransformant as described herein.

When a Microbacterium microorganism transformed with HRase is used, asubstrate may be added directly to a culture during or after culturingor any material containing the enzyme which includes, lysed or disruptedcells, crude enzyme preparations, enzyme preparations at any stage ofthe purfication process and/or purified enzyme.

In the process for producing an optically active amino acid according tothe present invention, the HRase of the present invention is combinedwith two other enzymes:(1) hydantoinase (Hhase), which hydrolyzes a5-substituted hydantoin compound to form N-carbamyl amino acid, and(2)N-carbamyl amino acid hydrolase (CHase), which hydrolyzes N-carbamylamino acid to form an optically active amino acid.

In the above, if the optical selectivity of HHase hydrolyzing a5-substituted hydantoin compound is high, either optically activeN-carbamyl-L-amino acid or N-carbamyl-D-amino acid can be formed inhigher yields (50% or more molar yield by the action of HRase) byallowing the HHase having high optical selectivity and the HRase of thepresent invention to act on the 5-substituted hydantoin. In this case,CHase or a material containing the enzyme may be subsequently used toproduce the optically active amino acid, or the optically active aminoacid can be produced in higher yields while maintaining optical activityby chemical hydrolysis treatment with nitrite (combination of microbialenzyme and chemical reaction systems).

HHase as used for optico-selective hydrolyses of 5-substituted hydantoincompounds can be obtained in the following manner. For example, thepresence of a thermostable D-HHase enzyme in Bacillus microorganismshaving D-HHase forming N-carbamyl-D-amino acid is known; for example,HHase or an HHase-containing fraction may be prepared from Bacillusstearothermophilus ATCC 31195. (Appl. Microbiol. Biotechnol., 43, 270(1995)), ATCC 31195 has been deposited at the American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md. 20852, United States ofAmerica. Further, the presence of L-HHase acting specifically onL-hydantoin compounds is known in e.g. Bacillus sp. AJ12299 (JapanesePatent Application Laid-Open (JP-A) No. 63-24894). Bacillus sp. AJ12299is a microorganism deposited on July 5, 1986 under FERM-P8837 with theNational Institute of Bioscience and Human-Technology, the Agency ofIndustrial Science of Technology, the Ministry of International Tradeand Industry.

Even if D- has no optico-selective hydrolysis activity, the formed aminoacid will be a D- or L-optically active isomer insofar as CHase hasoptical selectivity. In this case, decomposition of N-carbamyl-L-aminoacid by Chase to form and L- amino acid, N-carbamyl-D-amino acid willremain in the reaction solution, as wells as when D-amino acids areformed. When this occurs, Hhase will catalyze the reverse reation ofdehydrating and condensing N-carbamyl amino acids as the remainingunreacted enantioner, albeit at a lower rate than the forward reaction.This reverse reaction forms the 5-substituted hydantoin compound again,and therefore the optically active amino acid can be produced in highyield (at least 50% molar yield by the action of HRase) by the threeenzymes, that is, HRase, HHase and highly optically selective CHase, ormaterial(s) containing the three enzymes.

The presence of HHase having no optical selectivity in e.g. Arthrobacteraurescens, besides Microbacterium liquefaciens AJ3912 shown in thepresent invention, is known (J. Biotechnol. 61, 1 (1998)).

The presence of CHase selectively hydrolyzing a D-isomer of N-carbamylamino acid in e.g. Pseudomonas sp. AJ 11220 is known (Japanese PatentApplication Publication (JP-B) No. 56-003034). As a result of ourre-classification, Psudomonas sp. AJ1122—is classified as Agrobacteriumsp. Agrobacterium sp. AJ 11220 is a microorganism deposited on Dec. 20,1977 under FERM-P4347 with the National Institute of Bioscience andHuman-Technology, the Agency of Industrial Science of Technology, theMinistry of International Trade and Industry. Further, the presence ofCHase selectively hydrolyzing an L-isomer of N-carbamyl amino acid ine.g. Flavobacterium sp. AJ3912 (Japanese Patent Application Publication(JP-B) No. 56-008749) and Bacillus sp. AJ12299 is known. As describedabove, Flavobacterium sp. AJ3912, which is now classified intoMicrobacterium liquefaciens AJ3912 (FERM-P3133), is a microorganismdeposited on Jun. 27, 1975 under FERM-P3133 with the National Instituteof Bioscience and Human-Technology, the Agency of Industrial Science ofTechnology, the Ministry of International Trade and Industry. Further,Bacillus sp. AJ12299 is a microorganism deposited on Jul. 5, 1986 underFERM-P8837 with the National Institute of Bioscience andHuman-Technology, the Agency of Industrial Science of Technology, theMinistry of International Trade and Industry.

The substrate of HRase or a fraction containing HRase can make any5-substituted hydantoin compound which can be racemized with thesubstrate specificity of the enzyme. Examples of the 5-substitutedhydantoin compound include 5-indolyl methyl hydantoin,5-(p-hydroxybenzyl) hydantoin, 5-isobutyl hydantoin,5-(3′-pyridyl)-methyl hydantoin and other similar hydantoin compounds.

A preferable combination of HRase, HHase and CHase includes acombination of: HRase having the amino acid sequence of SEQ ID NO:2,HHase having the amino acid sequence of SEQ ID NO:4, and CHase havingthe amino acid sequence of SEQ ID NO:6. These enzymes are preferablyderived from Microbacterium liquefaciens AJ3912.

When HRase having the amino acid sequence set forth in SEQ ID NO:2 inthe Sequence Listing, HHase having the amino acid sequence in SEQ IDNO:4 in the Sequence Listing and CHase having the amino acid sequence inSEQ ID NO:6 in the Sequence Listing are used in combination, mixedproteins consisting of HRase, HHase and CHase, obtained by culturingcells transformed with a recombinant DNA comprising a vector ligated toa group of structural genes set forth in SEQ ID NO:7 coding for all theproteins involved in producing L-amino acid can also be used. If themixed proteins are used, the hydantoin racemase contained in the mixedproteins catalyses racemization of the 5-substituted hydantoin compoundas shown in the reaction scheme (II) below, so that from theDL-5-substituted hydantoin compound, L-amino acid can be produced intheoretically 100% molar yield.

The above mixed proteins can also be used to produce N-carbamyl aminoacids. For example, N-carbamyl amino acid can be produced by adding e.g.an inhibitor of the N-carbamyl-L-amino acid hydrolase to terminate thehydrolysis reaction at the stage of N-carbamyl amino acid.

When the HRase on any of the forms described herein is used in the aminoacid forming reaction the reaction solution is preferably adjusted to asuitable temperature of from about 25 to about 40° C. is incubated forabout 8 hours to about 5 days while maintaining the pH of the solutionat 5 to 9. The incubation can performed with or without stirring,shaking or agitation. Preferably, the culture is performed under aerobicconditions.

When the amino acid forming reaction is performed in culture, thewater-soluble medium used in the culture would contain a 5-substitutedhydantoin compound and nutrients such as a carbon source, a nitrogensource and inorganic ions necessary for growth of the transformed cellsas described herein. Preferably, the culture medium also containsorganic trace nutrients such as vitamins, amino acids, and othernutrients that facilate cell growth and expression of the enzymes. The5-substituted hydantoin compound may be added to the culture medium allat the same time, at the start of the culture or at some point during orafter the culturing, the 5-substituted hydantoin compound may be addedgradually to the culture, continously, in a gradient (increasingconcentrations or decreasing concentrations) or batch-wise to theculture.

The formed amino acid can be separated and purified by techniques knownin the art. Examples of such separation and purification protocolsinclude adsorbing the basic amino acid by contacting with anion-exchange resin, then eluting it followed by crystallization thereof,a method of discoloring the amino acid by filtration with activatedcarbon and then crystallizing it, etc. Other appropriate protocols canbe employed.

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples, which areprovided herein for purposes of illustration only, and are not intendedto be limiting unless otherwise specified.

EXAMPLES Example 1 Production and Purification of HRase

Microbacterium liquefaciens AJ3912 (FERM-P3133) was cultured to give themicroorganism having a sufficient HRase activity.

First, the microorganism was refreshed at 30° C. for 24 hours in CM2Gagar medium (0.5 g/dl D-glucose, 1 g/dl yeast extract, 1 g/dl peptone,0.5 g/dl NaCl, 2 g/dl agar, pH 7.0), followed by seed culture in CM2Gliquid medium at 30° C. for 24 hours.

Thereafter, 1 ml seed culture liquid was inoculated into a 500-mlSakaguchi flask containing 50 ml main culture medium and cultured undershaking at 30° C. for 18 hours. The main culture medium was prepared byautoclaving a medium of 0.5 g/dl D-glucose, 0.5 g/dl (NH₄)₂SO₄, 1 g/dlyeast extract, 1 g/dl peptone, 0.1 g/dl KH₂PO₄, 0.3 g/dl K₂HPO₄, 0.01g/dl MgSO₄.7H₂O, 0.35 g/dl DL-5-indolyl methyl hydantoin (IMH), pH 7.0,at 120° C. for 20 minutes, and then adding 1 mg/dl each of FeSO₄.7H₂O,MnSO₄.4-5H₂O, and CaCl₂.2H₂O thereto and leaving the medium at 4° C. toprecipitate IMH sufficiently.

After culture, the microorganism was collected by centrifugation, washedwith 0.1 M KPB (pH 7.0) to give the washed microorganism.

The microorganism thus obtained was used as a material from which HRasewas to be purified.

1. Disruption of the Microorganism

66 g of the washed microorganism (corresponding to 3.7 L culture liquid)was used as the starting material, and this microorganism was suspendedin 130 ml of 0.1 M KPB (pH 7.0) and disrupted with 0.1 mmφ glass beadsfor 3 minutes (30 seconds×6 times at 90-second intervals) with a beadbeater. The solution was recovered and treated with DNase I at a finalconcentration of 5 μg/ml at room temperature for 20 minutes. Thereafter,the undisrupted cells were removed by centrifugation at 13,000 g×10minutes, and the membrane fraction was further removed byultracentrifugation at 100,000 g×60 minutes, and the supernatant wasused as a cell-free extract.

2. Ammonium Sulfate Fractionation

59 g ammonium sulfate was added at a final concentration of 70%saturation to 125 ml of the cell-free extract, and the extract wasadjusted to pH 7.0 with KOH and stirred at 5° C. for 60 minutes. Theprecipitates were recovered by centrifugation at 13,000 g×15 minutes,dissolved in a small amount of 20 mM KPB (pH 7.0) and dialyzed against1.2 M (NH₄)₂SO₄, 20 mM KPB, 0.5 mM CoCl₂, pH 7.0 (Buffer A). Afterdialysis, the dialyzate was centrifuged at 13,000 g×30 minutes, and theresulting supernatant, 40 ml, was used in the following purification.

3. Hydrophobic Chromatography

The resulting enzyme solution was subjected to a hydrophobicchromatography column Phenyl Superose HP 26/10 (Pharmacia) previouslyequilibrated with Buffer A. The non-adsorbed protein was eluted withBuffer A, and then the adsorbed protein was eluted with a lineargradient (−1.2 M/12 CV) of from 1.2 to 0 M ammonium sulfate. When theHRase activity of each eluted fraction was measured, the activity wasrecognized in elution positions of from about 500 mM to 100 mM ammoniumsulfate. Fractions with the HRase activity were recovered, concentratedthrough a membrane and dialyzed against 20 mM KPB (pH 7.0).

4. Anion-Exchange Chromatography

The resulting enzyme solution was applied onto an anion-exchangechromatography column Q-Sepharose HP 16/10 (Pharmacia) previouslyequilibrated with 20 mM KPB (pH 7.0). After the non-adsorbed protein waseluted with 20 mM KPB (pH 7.0), the adsorbed protein was eluted with alinear gradient (0.5 M/12 CV) of from 0 to 0.5 M NaCl. When the HRaseactivity of each eluted fraction was measured, the activity wasrecognized in elution positions of from about 300 mM to 400 mM NaCl.Fractions with the HRase activity were recovered and concentratedthrough a membrane.

5. Gel Filtration

The resulting enzyme solution was applied onto Superdex 200 pg 16/60(Pharmacia) previously equilibrated with 20 mM KPB (pH 7.0), and thesample was developed with the same buffer. When the HRase activity ofeach eluted fraction was measured, the activity was recognized at theposition of an estimated molecular weight of about 107,0000. Fractionswith the HRase activity were recovered, concentrated through a membraneand dialyzed against 1.0 M (NH₄)₂SO₄, 20 mM KPB, 1 mM L-benzyl hydantoin(BH), pH 7.0 (Buffer B).

6. Hydrophobic Chromatography

The resulting enzyme solution was applied onto Phenyl Superose 5/5(Pharmacia) previously equilibrated with Buffer B. The non-adsorbedprotein was eluted with Buffer B, and then the adsorbed protein waseluted with a linear gradient (−1.2 M/17 CV) of from 1.2 to 0 M ammoniumsulfate. The HRase activity of each eluted fraction was measured, andfractions with the HRase activity were recovered and concentratedthrough a membrane.

Thereafter, this purification step using Phenyl Superose was performedonce in an L-BH-free buffer system and then once in an L-BH-containingbuffer system.

The enzyme solution obtained in the above procedures was used aspurified HRase solution.

The increase in specific activity brought about by the abovepurification was measured. By measuring the HRase activity of both thecell-free extract after disruption of the microorganism and the activefraction obtained after purification, it was found that the specificactivity per unit weight of the protein was increased 658-fold. In theactivity measurement method described later, the specific activity ofthe purified HRase was estimated to be 79 U/mg.

The resulting active fraction was subjected in a usual manner toSDS-polyacrylamide gel electrophoresis and stained with CoomassieBrilliant Blue, and as a result it was confirmed that the HRase waspurified to a single band, and its molecular weight was estimated to be27,000. In consideration of the result of gel filtration, it wasestimated that this HRase has a tetramer structure of subunits eachhaving a molecular weight of 27,000.

7. Determination of an Amino Acid Sequence in the Vicinity of theN-terminus

A sequence in the vicinity of the N-terminus of the HRase purified inthe manner described above was determined in the following manner.

That is, about 10 μg protein out of the purified HRase fraction wassubjected to polyacrylamide gel electrophoresis in the presence of SDS,and then the desired enzyme was transferred from the electrophoresed gelonto a polyvinylidene fluoride (PVDF (Bio-Rad, Trans-Blot)) membrane byuse of a Milliblot (Millipore) in a semi-dry system (“Tanpakushitsu KozoKaiseki” (Structural Analysis of Protein) written by Hisashi Hirano,Tokyo Kagaku Dojin). Subsequently, the desired enzyme on the PVDFmembrane was subjected to a protein sequencer (model 476A manufacturedby ABI), to analyze its N-terminal amino acid sequence.

The amino acid sequence of 30 residues from the N-terminus wasdetermined. The determined amino acid sequence in the vicinity of theN-terminus of HRase is set forth in SEQ ID NO:8 in the Sequence Listing.

Example 2 Effect of pH on HRase

The change (optimum pH) of the enzyme activity by reaction pH wasdetermined in the following manner.

0.3 μg/ml purified HRase was used and reacted at 37° C. for 30 minutesin the presence of 0.1 M sodium acetate buffer (pH 3.1, 3.9, 4.9, 6.1),KPB (pH 6.4, 7.2, 8.0), or sodium carbonate buffer (pH 8.2, 9.1, 10.2,10.9), to determine the reaction optimum pH of the HRase.

The measurement results were shown as relative enzyme activity againstthe actually measured pH of each reaction solution. For conveniencesake, the highest activity was given 100. The measurement results areshown in FIG. 3.

The optimum pH of the HRase of the present invention is in the pH rangeof about 7 to about 9, strictly about pH 8.0 to 9.0 (see FIG. 3).

Example 3 pH Stability of HRase

3.0 μg/ml purified HRase was left at 0° C. for 30 minutes in thepresence of 0.1 M buffer at each pH, and then adjusted to pH 8.0, andthe enzyme was subjected to reaction at pH 8.0, at 37° C. for 30minutes, and the residual activity was measured to determine the pHstability of the HRase.

The measurement results were shown as relative enzyme activity againstpretreatment at each pH. For convenience sake, the highest activity wasgiven 100. The measurement results are shown in FIG. 4.

It was found that the HRase of the present invention is stable in therange of about pH 6 to 9 (see FIG. 4).

Example 4 Effect of Temperature on HRase

0.3 μg/ml purified HRase was used and reacted at pH 8.0 for 30 minutesat 0, 21, 30, 40, 50, 60, 70 and 80° C. respectively, to determine theworking optimum temperature of the HRase.

The measurement results were shown as relative enzyme activity againstthe actually measured temperature of each reaction solution. Forconvenience sake, the highest activity was given 100. The measurementresults are shown in FIG. 5.

It was found that the optimum temperature of the HRase of the presentinvention is in the range of 50 to 60° C. (see FIG. 5). This optimumtemperature in reaction is the highest among known HRases.

Example 5 Temperature Stability of HRase

3.0 μg/ml purified HRase was left at pH 8.0 for 30 minutes at 0, 21, 30,40, 50, 60, 70 and 80° C., and then the enzyme was subjected to reactionat pH 8.0, at 37° C. for 30 minutes, and the residual activity wasmeasured to determine the temperature stability of the HRase.

The measurement results were shown as relative enzyme activity againstpretreatment at each temperature. For convenience sake, the highestactivity was given 100. The measurement results are shown in FIG. 6.

It was found that the HRase of the present invention is stable in therange of 40° C. or less (see FIG. 6).

Example 6 Effect of Various Reagents Such as Enzyme Inhibitors on HRaseActivity

1.5 μg/ml purified HRase was pre-incubated at 0° C. for 30 minutes at pH8.0 with 25 mM DTT, 25 mM N-ethylmaleimide (NEM), 12.5 mM CuSO₄, 50 mMmonoiodoacetic acid (IAA), 50 mM EDTA or 50% (v/v) methanol. As theenzyme source, each of these mixtures was reacted at pH 8.0, at 30° C.for 30 minutes to measure the residual activity, and the susceptibilityof the HRase to each inhibitor was determined. The concentration of eachof the enzyme and the inhibitor during the reaction was about 1/5relative to the concentration during the pre-incubation.

The residual activity was shown as relative activity to the activity(100%) in the absence of the reagent (see Table 2). The HRase of thepresent invention was activated by DTT (not shown in the table) andsignificantly inhibited by a cysteine residue-modifying reagent such asNEM, iodoacetic acid or Cu²⁺, suggesting the contribution of a cysteineresidue to expression of the activity. Because no significant inhibitoryeffect was observed when EDTA was added, it was considered that nodivalent ion is necessary for expression of the HRase activity. Further,pre-incubation with 50% (v/v) methanol did not influence the HRaseactivity (Table 2). TABLE 2 Reagent Concentration Residual Activity (%)None — 100 NEM  5 mM 0 CuSO₄ 2.5 mM  0 IAA 10 mM 0 EDTA 10 mM 86Methanol 10% (v/v) 99

Example 7 Racemization of BH by Purified HRase

A reaction solution containing 0.3 mg/ml purified HRase, 0.12 g/dl D- orL-BH, 50 mM KPB (pH 8.0) and 5 mM DTT was incubated at 37° C. andsampled with time to quantify the amount of BH by HPLC. As the control,the same experiment was performed in an experimental group notcontaining the enzyme, to quantify the spontaneous racemization of BH.The results are shown in FIG. 7.

The HRase of the present invention recognized both D-BH and L-BH assubstrates, and whichever substrate was used as the starting substrate,the reaction proceeded until the D, L-isomer ratio reached 1:1. However,the initial reaction rate of the racemization reaction was higher whenL-BH was used as the starting substrate, and the specific activity ofthe purified HRase as calculated from the initial reaction rate wasestimated to be 100 U/mg for formation of D-BH from L-BH and 79 U/mg forformation of L-BH from D-BH (see FIG. 7).

On the other hand, even in the control group where HRase was not added,spontaneous racemization of BH was observed, but the rate of thisracemization was significantly low as compared with the rate ofracemization catalyzed by HRase (see FIG. 7).

Example 8 Production of D-Phe from DL-BH or L-BH by a Combination ofAgrobacterium sp. AJ11220 and HRase

Agrobacterium sp. AJ11220 is a microorganism deposited on Dec. 20, 1977under FERM-P4347 with the National Institute of Bioscience andHuman-Technology, the Agency of Industrial Science of Technology, theMinistry of International Trade and Industry. The microbial strain isknown to hydrolyze a D-5-substituted hydantoin compound into itscorresponding D-amino acid (Agric. Biol. Chem., 51, 721 (1987)).However, AJ11220 does not have any hydantoin racemase so that unless thehydantoin compound spontaneously racemizes, the L-5-substitutedhydantoin compound not forming D-amino acid will remain, and therefore,the molar yield of D-amino acid formed from the DL-5-substitutedhydantoin compounds that can be prepared inexpensively by chemicalsynthesis is 50% at the maximum. Accordingly, the production of D-Phe bya combination of the AJ11220 microorganism and the purified HRase wasexamined using the substrate BH that could hardly spontaneouslyracemize.

1. Culturing of AJ 11220 Strain

The microorganism was refreshed at 30° C. for 24 hours in GM2G agarmedium (0.5 g/dl D-glucose, 1 g/dl yeast extract, 1 g/dl peptone, 0.5g/dl NaCl, 2 g/dl agar, pH 7.0), followed by seed culture in GM2G liquidmedium at 30° C. for 24 hours.

The main-culture medium used was prepared by autoclaving a mediumconsisting of 0.5 g/dl D-glucose, 0.5 g/dl (NH₄)₂SO₄, 1 g/dl yeastextract, 0.1 g/dl KH₂PO₄, 0.3 g/dl K₂HPO₄, 0.01 g/dl MgSO₄.7H₂O, pH 7.0,at 120° C. for 20 minutes and then adding 1 mg/dl each of FeSO₄.7H₂O andMnSO₄.4-5H₂O and 2 g/dl CaCO₃ thereto. 50 ml of this main-culture mediumwas introduced into a 500-ml Sakaguchi flask, and 1 ml of the seedculture liquid was inoculated thereinto and subjected to shake cultureat 30° C. for 22 hours. For inducing the enzyme, D-glucose and DL-BHwere added at final concentrations of 2 g/dl and 0.2 g/dl respectivelyin 14 hours after the main culture was initiated, and DL-BH was added ata final concentration of 0.2 g/dl in 16 and 18 hours after the mainculture was initiated.

After the culture was finished, the microorganism was collected bycentrifugation (10,000×g, 4° C., 10 minutes) and washed with 0.1 M KPB(pH 7.0), to give the washed microorganism.

2. Production of D-Phe form DL-BH or L-BH by a Combination of AJ 11220and HRase

0.5 g/dl (26 mM) DL-BH or L-BH, together with 0.84 μg/ml purified HRase,0.1 M KPB (pH 8.0), 0.5 mM CoCl₂ and 2 g/dl wet AJ11220 microorganism,was subjected to stationary reaction at 30° C. Oxygen was previouslyremoved from the reaction solution by replacing it by nitrogen. Thereaction solution was sampled with time and analyzed by HPLC to quantifythe formed D-Phe.

The time course of D-Phe formation is shown in FIG. 8. In the controlwhere D-BH was used as the substrate in the absence of HRase, almostevery substrate was converted into D-Phe in the reaction for 30 hours(amount of D-Phe produced, 25.4 mM and molar yield, 98% in □ in FIG. 8).On the other hand, the amount of D-Phe formed from DL-BH was 12.9 mM atthe maximum (reaction, 22 hours and molar yield, 49% in ◯ in FIG. 8),while the amount of D-Phe formed from L-BH was limited to 1.5 mM(reaction, 30 hours and molar yield, 6% in Δ in FIG. 8). The amount ofD-Phe formed from DL-BH as the substrate was about half of the amount ofD-Phe formed from D-BH as the substrate, and D-Phe was hardly formedfrom L-BH as the substrate, thus indicating that the rate-determiningreaction of forming D-Phe from BH in this reaction system is thereaction of racemizing L-BH into D-BH.

When HRase was added, on the other hand, it was observed that in thereaction for 30 hours, 24.6 mM D-Phe (molar yield, 94%) (▪ in FIG. 8)was formed from DL-BH, and 24.6 mM D-Phe (molar yield, 94%) (▴ in FIG.8) was formed from L-BH. These values were almost equivalent to thoseachieved by using D-BH as the substrate, suggesting that therate-determining reaction of racemizing L-BH into D-BH was almostcompletely resolved by adding HRase.

From the foregoing, when the DL- or L-5-substituted hydantoin compoundis allowed to act in the D-isomer-selective hydrolysis reaction systemto form its corresponding D-amino acid, in the case of 5-substitutedhydantoin compound used as the substrate having no or less spontaneousracemization ability, the L-5-substituted hydantoin compound notundergoing hydrolysis will remain unreacted in the reaction solution;however, by combining the reaction system with HRase, the whole of thesubstrate can be efficiently hydrolyzed to form its correspondingD-amino acid in higher yield.

As a matter of course, when the 5-substituted hydantoin compound isallowed to act similarly in the L-isomer-selective hydrolysis reactionsystem to form its corresponding L-amino acid, the whole of thesubstrate can be efficiently hydrolyzed by combining the reaction systemwith HRase, whereby its corresponding L-amino acid can be formed inhigher yield.

Example 9 Racemization of Various Optically Active 5-substitutedHydantoin Compounds Other Than BH

The ability of the present HRase to catalyze racemization of opticallyactive 5-substituted hydantoin compounds other than BH was examined. Theformation of D-amino acid from L-5-substituted hydantoin by acombination of Pseudomonas sp. AJ 11220 and HRase was confirmed in thesame manner as in Example 8.

1 g/dl 5-substituted hydantoin of various kinds, together with 1.3 μg/mlpurified HRase, 0.1 M KPB (pH 8.0), 0.5 mM CoCl₂ and 2 g/dl wet AJ11220, was subjected to stationary reaction at 37° C. for 22 hours. Eachhydantoin consisted of D- and L-isomers as the substrate, and it wasconfirmed that D-amino acid was formed from the D-isomer substrate inthe absence of HRase, and also that D-amino acid was not formed from theL-isomer substrate in the absence of HRase, and then it was judged thatwhen D-amino acid was formed from the L-isomer substrate in the presenceof HRase, the substrate was recognized. The formation of D-amino acidwas qualitatively estimated by development with optical resolution TLC(MERCK, HPTLC CHIR) with methanol: H₂O acetonitrile=1:1:4.

As a result, it was recognized that HRase racemized L-5-indolyl methylhydantoin, L-5-(p-hydroxybenzyl) hydantoin and L-5-isobutyl hydantoin toform their corresponding D-tryptophan, D-tyrosine and D-leucinerespectively.

Example 10 Isolation of HRase Gene

Hereinafter, isolation of HRase gene and expression of HRase in E. coliare described where Microbacterium liquefaciens AJ3912 (FERM-P3133) wasused as the bacterial strain. Isolation of the gene and expression ofHRase were conducted using E. coli JM109 as the host and pUC18 as thevector.

1. Preparation of PCR Primers Based on the Determined Amino AcidSequence

On the basis of the N-terminal amino acid sequence (SEQ ID NO:8 in theSequence Listing) of HRase of Microbacterium liquefaciens AJ3912(FERM-P3133), mix primers shown SEQ ID NOS:9 and 10 respectively wereprepared.

2. Acquisition of the Microorganism

Microbacterium liquefaciens AJ3912 (FERM-P3133) was refreshed byculturing it at 30° C. for 24 hours in CM2G agar medium (0.5 g/dlglucose, 1.0 g/dl yeast extract, 1.0 g/dl peptone, 0.5 g/dl NaCl, 2 g/dlagar, pH 7.0). The microorganism was inoculated via one loop of platinuminto a 500-ml Sakaguchi flask containing 50 ml CM2G liquid medium, andthen cultured under shaking at 30° C. for 16 hours under aerobicconditions.

3. Acquisition of the Chromosomal DNA from the Microorganism

50 ml of the culture liquid was centrifuged (12,000×g, 4° C., 15minutes), whereby the microorganism was collected. This microorganismwas suspended in 10 ml of 50:20 TE (50 mM Tris-HCl, pH 8.0, 20 mM EDTA),washed and centrifuged, whereby the microorganism was recovered. Thismicroorganism was suspended again in 10 ml of 50:20 TE. 0.5 ml of 20mg/ml lysozyme solution and 1 ml of 10% SDS solution were added to theabove suspension, followed by incubation at 55° C. for 20 minutes. Afterincubation, a 1-fold volume of 10:1 TE-saturated phenol was addedthereto to remove proteins. A 1-fold volume of 2-propanol was added tothe separated aqueous layer, to precipitate and recover DNA. After theprecipitated DNA was dissolved in 0.5 ml of 50 : 20 TE, 5 μl of 10 mg/mlRNase and 5 μl of 10 mg/ml Proteinase K were added thereto and themixture was reacted at 55° C. for 2 hours. After reaction, a 1-foldvolume of 10:1 TE-saturated phenol was added thereto to remove proteins.Further, a 1-fold volume of 24:1 chloroform/isoamyl alcohol was addedthereto and stirred, and the aqueous layer was recovered. This procedurewas repeated further twice, and 3 M sodium acetate solution (pH 5.2) wasadded at a final concentration of 0.4 M to the resulting aqueous layer,followed by adding a 2-fold volume of ethanol thereto. The DNA occurringas precipitates was recovered, washed with 70% ethanol, dried anddissolved in 1 ml of 10:1 TE.

4. Acquisition of a DNA Fragment Containing a Part of the HRase Gene bythe Cassette PCR Method

For isolation and amplification of the DNA molecule containing the gene(fhr) coding for HRase by the cassette PCR method, TaKaRa LA PCR invitro Cloning Kit (Takara Shuzo Co., Ltd) was used. Unless otherwisenoted, the experiment was carried out according to instructions of thekit. In the cassette PCR method, when primer 1 (1st PCR) and primer 2(2nd PCR) were used as the primers, an about 0.4-kb band (fragment 1)was amplified with HindIII cassette. By determining the nucleotidesequence of this fragment, fragment 1 was confirmed to be a part of fhr.

5. Cloning of the HRase Gene From a Gene Library

Then, Southern hybridization with fragment 1 as a probe was carried outto obtain the full-length fhr.

The concentration of the DNA fragment serving as a probe was adjusted toabout 50 ng/μl, and 16 μl of this DNA solution was labeled as the probeby incubating it at 37° C. for 24 hours with DIG High Prime (BoehringerMannheim) according to its protocol.

1 μg chromosomal DNA was completely digested with a combination ofvarious restriction enzymes, subjected to electrophoresis on 0.8%agarose gel, and blotted onto a nylon membrane (Boehringer Mannheim,Nylon membranes positively charged). Subsequently, Southernhybridization was conducted according to the conventional method.Hybridization was carried out using DIG Easy Hyb (Boehringer Mannheim),and after pre-hybridization at 50° C. for 30 minutes, the probe wasadded and hybridized at 50° C. for 18 hours. The sample was detectedusing a DIG Nucleotide Detection Kit (Boehringer Manheim).

As a result, a band was detected at a position of about 2.9 kb in theproduct cleaved with EcoRI/PstI.

This 2.9-kb fragment was recovered and ligated to pUC18, to prepare alibrary (120 strains) with E. coli JM109. Subsequently, colonyhybridization was conducted according to the conventional method. Thecolonies were transferred onto a nylon membrane filter (BoehringerMannheim, Nylon membranes for colony and plaque hybridization), followedby alkali denaturation, neutralization and immobilization. Hybridizationwas conducted using DIG Easy Hyb. The filter was immersed in a bufferand subjected to pre-hybridization at 42° C. for 30 minutes. Thereafter,the above-described labeled probe was added thereto and hybridized at42° C. for 18 hours. After washing with SSC buffer, 1 positive clonestrain was selected by using a DIG Nucleotide Detection Kit. 6.Nucleotide sequence of the HRase gene derived from Microbacteriumliquefaciens AJ3912 (FERM-P3133) The plasmid maintained by the selectedtransformant was prepared by a method described in Molecular Cloning,2nd edition, Cold Spring Harbor Press (1989), and a nucleotide sequencenear to the sequence hybridized with the probe was determined. An openreading frame (ORF) coding for a protein containing the N-terminal aminoacid sequence of 30 residues in HRase was present therein and confirmedto be the gene fhr coding for HRase. The nucleotide sequence of thefull-length HRase gene is shown in SEQ ID NO: 1 in the Sequence Listing.The resulting ORF showed 48% homology to known HRases (J. Bacteriol.,174, 962 (1992)) derived from microorganisms of the genus Pseudomonas.

7. Expression of HRase gene in E. coli

To express fhr in E. coli, plasmid pUCFHR was constructed by ligatingfhr to a downstream region of a lac promoter in pUC18. PCR was performedwhere the chromosomal DNA from Microbacterium liquefaciens AJ3912(FERM-P3133) was used as the template and the oligonucleotides shown inTable 2 were used as the primers, and the amplified fragment was treatedwith EcoRI and BamHI and ligated to an EcoRI/BamHI-cleaved product ofpUC18, and then transformed into E. coli JM109. From ampicillinresistant strains, a strain having the desired plasmid was selected, andthe constructed plasmid was designated expression plasmid pUCFHR. TABLE3 Primer Sequence 5′-side GCCGAATTCGCGACTGGCAACACGAAGG SEQ ID NO:11     EcoRI 3′-side CGGGGATCCTTCTCGTTAGAGGTACTGC SEQ ID NO:12      BamHI

The HRase E. coli transformant harboring PUCFHR was subjected to seedculture at 37° C. for 16 hours in LB medium containing 0.1 mg/mlampicillin. 1 ml of this pre-culture liquid was seeded in a 500-mlSakaguchi flask containing 50 ml LB medium, and then cultured at 37° C.In 2.5 hours after culture was initiated,isopropyl-1-thio-β-D-galactopyranoside (IPTG) was add thereto at a finalconcentration of 1 mM, and culture was further continued for 4 hours.

After the culture was finished, the microorganism was collected, washed,suspended in 5 ml of 50 mM KPB (pH 8.0) and disrupted with 0.1 mmφ glassbeads for 3 minutes (30 seconds×6 times at 90-second intervals) with abeads beater. The solution was recovered and centrifuged at 20,000 g×10minutes, and the supernatant was used as the cell-free extract.

8. Measurement of HRase Activity

After the culture, the cell-free extract was prepared as an enzymesource and measured for its HRase activity.

For measurement of the HRase activity, a reaction solution containing120 mg/dl L-BH, 50 mM KPB (pH 8.0), 5 mM DTT, 5 mM EDTA, 150 mM NaCl andthe enzyme solution was incubated at 37° C. for 30 minutes, and thereaction was terminated by adding a 9-fold volume of 1.1 mM CuSO₄ and11.1 mM H₃PO₄. By centrifugation at 20,000 g×10 minutes, precipitateswere removed, and the amount of BH racemized was quantified by HPLC todetermine the HRase activity. The enzyme activity causing racemizationof 1 μmol BH per minute under these conditions was defined as 1 U of theenzyme activity.

The HPLC conditions used in analysis are as follows:

Column: Daicel Chemical CHIRALPAK WH 0.46 cmφ×25 cm

Mobile phase: 5% (v/v) methanol, 1 mM CuSO₄

Column temperature: 50° C.

Flow rate: 1.5 ml/min.

Detection: UV₂₁₀

As a result, the HRase activity was detected in only the E. coli havingpUC18FHR introduced therein, and it was confirmed that the cloned fhrgene was expressed in E. coli (Table 4). TABLE 4 Expression of HRase byE. coli having pUC18FHR introduced therein Addition Proteinconcentration HRase of (mg/ml) in the activity IPTG cell-free extract(U/mg) pUC18FHR + 1.9 0.15 pUC18FHR − 3.4 0.06 pUC18 + 2.2 Not detectedpUC18 − 3.7 Not detected

According to the present invention, HRase not known so far can beobtained from a microorganism of the genus Microbacterium. As comparedwith HRases known in the art, this HRase is characterized by a higheroptimum reaction temperature, so the reaction temperature can beincreased, resulting in an increase in the reaction rate and efficientprogress of the desired reaction. Further, the risk of contamination ofthe reaction solution with microorganisms during the reaction can bereduced and thus there is the advantage of easy process controlincluding quality control. Further, by using the hydantoin racemase ofthe present invention in combination with a system of hydrolyzing5-substituted hydantoin compounds optico-selectively, optically activeamino acids useful for production pharmaceutical preparations, productsin chemical industry, food additives can be formed in higher yield.

The present application claims priority to JP2000-278571 filed Sep. 13,2000 and JP2001-65815 filed Mar. 8, 2001, the entire contents of bothapplications are incorporated herein by reference.

Obviously, numerous modifications and variations on the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

1. A method for producing an N-carbamyl amino acid, comprisingcontacting a 5-substituted hydantoin with an isolated polypeptidecomprising the amino acid sequence of SEQ ID NO:2 and a isolatedhydantoinase protein.
 2. The method of claim 1, wherein said contactingis performed in a culture comprising a microorganism expressing theisolated polypeptides.
 3. The method of claim 1, wherein said isolatedpolypeptides are in a cell lysis preparation.
 4. The method of claim 1,wherein said 5-substituted hydantoin compound is at least one compoundselected from the group consisting of include 5-indolyl methylhydantoin, 5-(p-hydroxybenzyl) hydantoin, 5-isobutyl hydantoin, and5-(3′-pyridyl)-methyl hydantoin.
 5. The method of claim 1, wherein theisolated hydantoinase protein comprises the amino acid sequence of SEQID NO:4.
 6. A method for producing an N-carbamyl amino acid, comprisingcontacting a 5-substituted hydantoin with an isolated 5-substitutedhydantoin racemase, which racemizes 5-substituted hydantoin, wherein the5-substituted hydantoin racemase has an optimal working pH of from about7 to about 9 and an optimal working temperature of from about 50 toabout 60° C. and a isolated hydantoinase protein.
 7. The method of claim6, wherein said contacting is performed in a culture comprising amicroorganism expressing the isolated polypeptides.
 8. The method ofclaim 6, wherein said isolated polypeptides are in a cell lysispreparation.
 9. The method of claim 6, wherein said 5-substitutedhydantoin compound is at least one compound selected from the groupconsisting of include 5-indolyl methyl hydantoin, 5-(p-hydroxybenzyl)hydantoin, 5-isobutyl hydantoin, and 5-(3′-pyridyl)-methyl hydantoin.10. The method of claim 6, wherein the isolated hydantoinase proteincomprises the amino acid sequence of SEQ ID NO:4.
 11. A method ofproducing optically active amino acids, comprising contacting a5-substituted hydantoin with an isolated polypeptide comprising theamino acid sequence of SEQ ID NO:2 and a isolated hydantoinase toproduced a N-carbamyl amino acid; and contacting said N-carbamyl aminoacid with a carbamyl amino acid hydrolase.
 12. The method of claim 11,wherein said contacting is performed in a culture comprising amicroorganism expressing the isolated polypeptides.
 13. The method ofclaim 12, wherein the microorganim expressing the isolated polypeptidescomprises an isolated polynucleotide comprising the nucleotide sequenceof SEQ ID NO:7.
 14. The method of claim 11, wherein said isolatedpolypeptides are in a cell lysis preparation.
 15. The method of claim11, wherein said 5-substituted hydantoin compound is at least onecompound selected from the group consisting of include 5-indolyl methylhydantoin, 5-(p-hydroxybenzyl) hydantoin, 5-isobutyl hydantoin, and5-(3′-pyridyl)-methyl hydantoin.
 16. The method of claim 11, wherein theisolated hydantoinase protein comprises the amino acid sequence of SEQID NO:4.
 17. The method of claim 11, wherein the isolated carnamyl aminoacid hydrolase comprises the amino acid sequence of SEQ ID NO:6.
 18. Amethod of producing optically active amino acids, comprising contactinga 5-substituted hydantoin with an isolated 5-substituted hydantoinracemase, which racemizes 5-substituted hydantoin, wherein the5-substituted hydantoin racemase has an optimal working pH of from about7 to about 9 and an optimal working temperature of from about 50 toabout 60° C. and a isolated hydantoinase to produced a N-carbamyl aminoacid; and contacting said N-carbamyl amino acid with a carbamyl aminoacid hydrolase.
 19. The method of claim 18, wherein said contacting isperformed in a culture comprising a microorganism expressing theisolated polypeptides.
 20. The method of claim 19, wherein themicroorganim expressing the isolated polypeptides comprises an isolatedpolynucleotide comprising the nucleotide sequence of SEQ ID NO:7. 21.The method of claim 18, wherein said isolated polypeptides are in a celllysis preparation.
 22. The method of claim 18, wherein said5-substituted hydantoin compound is at least one compound selected fromthe group consisting of include 5-indolyl methyl hydantoin,5-(p-hydroxybenzyl) hydantoin, 5-isobutyl hydantoin, and5-(3′-pyridyl)-methyl hydantoin.
 23. The method of claim 18, wherein theisolated hydantoinase protein comprises the amino acid sequence of SEQID NO:4.
 24. The method of claim 18, wherein the isolated carnamyl aminoacid hydrolase comprises the amino acid sequence of SEQ ID NO:6.